Cutting Edge: Bispecific γδ T Cell Engager Containing Heterodimeric BTN2A1 and BTN3A1 Promotes Targeted Activation of Vγ9Vδ2+ T Cells in the Presence of Costimulation by CD28 or NKG2D.

Vγ9Vδ2+ T cell-targeted immunotherapy is of interest to harness its MHC-independent cytotoxic potential against a variety of cancers. Recent studies have identified heterodimeric butyrophilin (BTN) 2A1 and BTN3A1 as the molecular entity providing "signal 1" to the Vγ9Vδ2 TCR, but "signal 2" costimulatory requirements remain unclear. Using a tumor cell-free assay, we demonstrated that a BTN2A1/3A1 heterodimeric fusion protein activated human Vγ9Vδ2+ T cells, but only in the presence of costimulatory signal via CD28 or NK group 2 member D. Nonetheless, addition of a bispecific γδ T cell engager BTN2A1/3A1-Fc-CD19scFv alone enhanced granzyme B-mediated killing of human CD19+ lymphoma cells when cocultured with Vγ9Vδ2+ T cells, suggesting expression of costimulatory ligand(s) on tumor cells is sufficient to satisfy the "signal 2" requirement. These results highlight the parallels of signal 1 and signal 2 requirements in αß and γδ T cell activation and demonstrate the utility of heterodimeric BTNs to promote targeted activation of γδ T cells.

High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.

We have developed a high-throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following: (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4(+) T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6 ± 2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.

The role of CXCR4 in maintaining peripheral B cell compartments and humoral immunity.

The chemokine receptor CXCR4 is expressed in B cells at multiple stages of their development. CXCR4 function in humoral immunity has not been fully investigated. We have generated gene-targeted mice in which CXCR4 can be selectively inactivated in B cells and have shown that it is required for retention of B cell precursors in the bone marrow. CXCR4-deficient B cell precursors that migrated prematurely became localized in splenic follicles despite their unresponsiveness to CXCL13. Concomitantly, mature B cell populations were reduced in the splenic marginal zone and primary follicles, and in the peritoneal cavity in the mutant animals, as were T-independent antibody responses. In addition, aberrant B cell follicles formed ectopically in intestinal lamina propria around Peyer's patches. These findings establish an important role for CXCR4 in regulating homeostasis of B cell compartmentalization and humoral immunity.